Fatty aldehyde bisulfite adducts as a purification handle in ionizable lipid synthesis

Rapid access to ALC-0315, a crucial component of the formulated Pfizer Covid vaccine, was obtained by employing solid adduct formation and filtration after an oxidation step in place of the standard chromatographic separation, allowing for a more scalable synthesis. Impurities were removed by formation of this fatty aldehyde bisulfite adduct at the penultimate step and by performing the final reductive amination directly with the fatty aldehyde bisulfite adduct. This eliminates chromatographic separations for all prepared aldehyde containing intermediates. Along with ALC-0315, FTT5 and SM-102 ionizable lipids were prepared utilizing this strategy. This work paves the way for more sustainable access to these critical ionizable lipids that would de-risk the world supply of important vaccines and medicines in the future.


Introduction
Lipid nanoparticles (LNPs) play a crucial role in the formulation of nucleic acid derived medicines.2][3][4][5] These lipids are required in signicantly larger quantities than the nucleic acids they carry.However, their synthesis includes major drawbacks from a process chemistry perspective such as excessive use of chlorinated solvents and multiple column chromatography operations.ALC-0315 1 is the ionizable lipid in the Pzer-BioNTech Covid-19 vaccine, Comirnaty, (0.43 mg per dose) and an estimated 4.6 billion doses were shipped to 181 countries. 6Therefore, since 2020 more than 2 MT of ALC-0315 was produced.Herein, we present our effort to make ionizable lipid production more sustainable by developing a route that relies on bisulte adduct formation and ltration rather than chromatographic purication of intermediates to obtain analytically pure lipid.
][9][10] However, they all have signicant drawbacks from a process chemistry perspective, using skin sensitizing reagents, chlorinated solvents and having a low control on impurity management requires the use of column chromatography purication at multiple steps.To overcome these challenges and allow for a more sustainable and scalable synthesis, it was envisioned that the desired lipid could be obtained via a fatty aldehyde bisulte adduct intermediate 4 of the analogous aldehyde 3 which would itself act as a purication handle in the synthesis.Additionally, the bisulte adduct can be used directly in the next reductive amination step to produce 1.
To generate the required aldehyde 3, an acid-catalyzed esterication using the non-symmetrical 6-bromo-1-hexanol could be employed to avoid bis-ester formation.Whereas, prior reports relied on non-selective esterication of symmetrical diols, which typically requires the use of chromatographic separation of the undesired bis-ester byproduct or the use of a large molar excess of diol starting material relative to the carboxylic acid in order to disfavor bis-ester formation.The obtained bromo-ester 2 could then undergo a Ganem type oxidation to furnish the aldehyde 3. 11,12 This would allow the aldehyde 3 to be puried via bisulte adduct formation.Alternatively, the direct alkylation of the amine with the bromo-ester 2 to form ALC-0315 1 is known in the literature but produces the quaternary ammonium salt as a side-product which is not easily removed or controlled (Scheme 1, conditions b). 7 Finally, a modication of the known reductive amination would provide ALC-0315 1 by direct reaction of the fatty aldehyde bisulte adduct 4 in 2-MeTHF as an alternative to the commonly used CH 2 Cl 2 , increasing the selectivity without needing to add solubilizing groups such as silyl ethers (Scheme 1, conditions e) 8 which can increase step count as well as unit operations dramatically.[9][10] The strategy of purication via solid fatty aldehyde bisulte adduct followed by reductive amination directly with the bisulte adduct lends itself well to a large range of ionizable lipids due to structural similarities (Chart 1) and, therefore, retrosynthetic approaches.Select examples of other ionizable lipids (FTT5 5, 13 a lipid-like compound which has found in vivo success, and SM-102 6 from Moderna's mRNA-1273 COVID-19 vaccine) were synthesized to further demonstrate the utility of this synthetic strategy.
To the best of our knowledge, solid fatty aldehyde bisulte adduct intermediates have not been previously reported in the eld of ionizable lipid synthesis nor used in subsequent reactions directly.

Results
The initial step in the synthesis of ALC-0315 1 being an esteri-cation allows for a variety of potential reaction conditions to be utilized.][16][17] Using standard Dean-Stark azeotropic conditions the bromoester 2 is generated in good yield (96%) and purity (>95% by HPLC-CAD).
With the bromo-ester 2 in hand, subsequent oxidation to the aldehyde was developed, which was adapted from an analogous oxidation (see ESI † for more details). 12Optimization of this reaction (Table S3 †) culminates in bromide 2 being dosed into a reuxing solution of pyridine N-oxide, NaOAc, and n-propyl acetate over 4 hours to generate aldehyde 3 in good yield (88% NMR yield).
With general conditions for the alkyl bromide oxidation to an aldehyde, purication of the crude aldehyde 3 via bisulte adduct is seen as a means of not only overcoming impurities 18 generated in the developed oxidation but as a potentially more appropriate starting material for the nal reductive amination to generate 1. [19][20][21] This would also act as a more suitable hold point when compared to typical aldehyde stability. 22nitial attempts to purify the crude aldehyde 3 involved dissolution of the crude oil in EtOH.This solution is then subjected to an aqueous solution of sodium metabisulte dropwise.The bisulte adduct 4 forms within 30 minutes (monitored by 1 H NMR) but gives a sticky, gummy solid material which is not suitable for ltration.Aqueous extraction of the adduct is not feasible due to limited solubility.
Removal of the water that is introduced during bisulte adduct formation via azeotropic distillation with toluene gives a more workable solid aer addition of EtOH, but unfortunately is still unsuitable for ltration due to clogging.
The results of a stability test of the adduct 4 indicate complete degradation of adduct and loss of product in acetone at extended contact times.Changing from acetone to ethyl formate improves the stability of the bisulte adduct, as determined by 1 H NMR (Fig. S1 †), while still enabling ltration.This is likely due to the similar size and polarity of ethyl formate, while not reacting with the in situ generated bisulte anions as acetone does.Additionally, spiking experiments also indicate that water does not have an impact on bisulte stability (mechanistic detail Scheme S3 †).
Upon addition of ethyl formate to the solid bisulte material, a brown solution is obtained along with an easily lterable light-tan colored solid giving 23.86 g (73% yield) of 4 as a light-brown to tan solid from the starting bromo-ester 2. This tan solid is noted to be hygroscopic as before, and can be stored either under nitrogen, or in a desiccator.A signicant amount of semisolids are found to have collected on the underside of the lter paper, slowing ltration.This is likely due to the relatively low boiling point of ethyl formate and the low pressure experienced aer passing through the lter.A simple change to DMC negates this issue and allows for a more rapidly ltering mixture.
The isolated adduct 4 is readily converted to the free aldehyde via washing with 10% sodium carbonate solution and extracting into EtOAc.The free aldehyde product is obtained with good purity (85-88%, HPLC-CAD) and can be used in the nal reductive amination step.With the free aldehyde in hand from 4, the reductive amination to generate ALC-0315 1 is preformed via a dosed addition of the free aldehyde with portionwise addition of the reducing agent, NaBH(OAc) 3 .Portion-wise addition of the reducing agent is needed to overcome process limitations. 8These conditions give purity of ALC-0315 1 in the isolated crude oil of 68% as analyzed by HPLC-CAD.Alternatively, the reductive amination can be performed directly from the isolated solid bisulte adduct 4 (Scheme 1, conditions o) to give an improved crude purity (86% HPLC-CAD), while also allowing 2-MeTHF to replace CH 2 Cl 2 as the solvent.Column chromatography gives puried ALC-0315 1 as a clear slightly yellow oil (53% isolated yield, 94.9% HPLC-CAD).This represents a 37% overall yield from the starting hexyldecanoic acid.
The main impurities in the nal isolated ALC-0315 1 are two structurally related ether impurities, 7 and 8 (Chart 2), which stems from an ether impurity present in the 6-bromo-1-hexanol starting material.This ether impurity in the esterication starting material culminates in two structurally similar compounds to ALC-0315 which are not easily separated via column chromatography.Therefore, an alternative synthetic route was evaluated, avoiding 6-bromo-1-hexanol which contains the impurity.
A straightforward change of the rst two steps eliminated the dependence on the problematic starting material and avoids the structurally similar impurities, 7 and 8, albeit with the introduction of a coupling reagent.An initial EDC coupling of hexyldecanoic acid and 1,6-hexandiol gives the mono-esteried diol 9 in high yield and good purity (94%, 88.0%HPLC-CAD), with the main impurity being the bis-ester byproduct (9.5%), which can be easily removed in a later step during purication of the aldehyde bisulte-adduct.Alcohol-ester 9 is then oxidized to aldehyde 3 with PIDA in the presence of cat.5][26] The crude aldehyde 3 is then subjected to the general bisulte adduct purication conditions.This gives crystalline off-white solid which lters rapidly to give the puried product as the sodium bisulte adduct 4 (Fig. 1) in a 90% yield (83% purity by HPLC-CAD) from 9, and overall yield of 85% from hexyldecanoic acid.This solid isolate eliminates the need for a chromatographic purication prior to its use in the following step, signicantly reducing waste that is typically generated in such chromatographic operations (Table S4 †).The adduct 4, as before, is subjected to the improved reductive amination conditions and column puried to give ALC-0315 1 in a 64% yield for the nal reductive amination step and 54% overall yield of ALC-0315 1 from the starting material, hexyldecanoic acid.This yield is an improvement when compared to other literature preparations of ALC-0315 1 (20-47%, Scheme 1) 7-9 as well as patented procedures (50%). 10The culmination of the process yields ALC-0315 1 with a purity of 97.3% as analyzed by HPLC-CAD with no structurally similar impurities, 7 or 8, present.This material was subsequently used in a formulation preparation, encapsulating mRNA producing LNPs which match the physiochemical properties of formulation batches that utilize commercially available ALC-0315 (Table S6

†).
With the ability to synthesize ALC-0315 1 in this manner, FTT5 5, a proven lipid in vivo, 13 and SM-102 6 were the next targets for evaluating the route's feasibility across multiple lipids.
Preparation of the bisulte adduct intermediate 13 of FTT5 5 follows analogously to 4 (Schemes 2 and S1 † for full route) and gives a well behaving aky tan solid (Fig. 2).This, again, eliminates the need for a chromatographic separation of the aldehyde intermediate.Direct reaction of amine 10 with bisulte adduct 13 gives FTT5 5 (26%), closely matching the literature yield (27%). 13 The inclusion of IPA in the reaction is required to solubilize the amine 10, otherwise a very slow reaction is observed.
The synthetic route to SM-102 6 proceeds through bisulte adduct 19, aer oxidation of bromo-ester 18 (Schemes 3 and S2 † for full route).Isolation of the bisulte adduct 19 gives a well behaving off-white to tan solid (Fig. 3).Aer direct reaction of bisulte adduct 19 with amine 15 and column purication, SM-102 6 is obtained in good yield and purity (67% yield, 96.0% purity by HPLC-CAD).As with ALC-0315 1 and FTT5 5, SM-102 6 is readily prepared via a solid aldehyde bisulte adduct intermediate, enabling the elimination of chromatographic purication of the aldehyde intermediate, as well as the use of the bisulte adduct 19 directly in the nal step to produce the lipid.

Synthetic procedures
General procedure for synthesis of bromo-esters from bromo-alcohols.To a 3-neck RBF with attached Dean-Stark (open at top) and nitrogen inlet is added the acid (1 eq.), followed by toluene (5 V), bromo-alcohol (0.98 eq.), and p-TsA (0.1 eq.).This is brought up to a reux with a very slight nitrogen ow.Upon collection of distillates in the Dean-Stark, the Dean-Stark is lled to just below overow with toluene and the reaction is allowed to reux for 16 hours or until the reaction is complete via 1 H NMR before it is cooled to room temperature.The reaction mixture is transferred to a separatory funnel and the RBF is washed with 5 V sat.NaHCO 3 .This is transferred into the separatory funnel, shaken, and allowed to separate.The organic layer is separated, dried (Na 2 SO 4 ) and concentrated to give a slightly yellow to light brown oil.
General procedure for synthesis of bromo-esters from bromo-acids.To a 3-neck RBF with attached Dean-Stark (open at top) and nitrogen inlet is added the bromo-acid (0.98 eq.), followed by toluene (5 V), alcohol (1 eq.), and p-TsA (0.1 eq.).This is brought up to a reux with a very slight nitrogen ow.Upon collection of distillates in the Dean-Stark, the Dean-Stark is lled to just below overow with toluene and the reaction is allowed to reux for 16 hours or until the reaction is complete via 1 H NMR before it is cooled to room temperature.The reaction mixture is transferred to a separatory funnel and the RBF is washed with 5 V sat.NaHCO 3 .This is transferred into the separatory funnel, shaken, and allowed to separate.The organic layer is separated, dried (Na 2 SO 4 ) and concentrated to give a slightly yellow to light brown oil.
General procedure for synthesis of fatty aldehyde bisulte adducts.To a RBF with attached condenser is added pyridine Noxide (6.0 eq.), n-propyl acetate (9 V), and sodium acetate (2.0 eq.).This is brought to a reux before the bromo-ester is added, as a solution in n-propyl acetate (1 V), to the reuxing solution over 4 hours.Aer addition is complete the reaction is allowed to reux for a further 16 hours before cooling to room temperature.To the cooled mixture is added HPW (10 V).This is shaken, separated and organic is washed with sat.NaHCO 3 (10 V) before drying over Na 2 SO 4 and ltering.To the n-propyl acetate solution at 35 °C is added sodium metabisulte as a solution in water (0.45 g mL −1 ) dropwise.This is allowed to stir at 35 °C for 30 minutes before concentrating in vacuo.Aer concentrating an additional 10 V n-propyl acetate is charged and concentrated giving a solid material.To the solid is added ethyl formate or DMC (5 V) and the mixture is stirred at room temperature until all the solid material has formed a white to tan solid suspension in the mixture.This mixture is ltered, washed with ethyl formate or DMC (2 V) and the solids are collected and dried in a vacuum oven for 16 hours with a slight nitrogen bleed before weighing the collected bisulte adduct.
General procedure for synthesis of ionizable lipids via reductive amination of fatty aldehyde bisulte adducts.To a RBF with attached nitrogen inlet is added bisulte adduct (2.3 eq.), and 2-MeTHF (10 V to the adduct) at RT.This is allowed to stir until a homogeneous mixture is observed.NEt 3 (2.4 eq.) is added to the reaction followed by the reactive amine (1 eq.) and then NaBH(OAc) 3 (4.3eq.).The reaction is stirred for 16 h at RT before the reaction solution is washed with sat.Na 2 CO 3 (10 V to adduct).The organic layer is dried (Na 2 SO 4 ), ltered, and concentrated before being puried via column chromatography (SiO 2 ).

Conclusions
A general route has been developed for multiple commonly used ionizable lipids which have been prepared through solid fatty aldehyde bisulte adduct intermediates, eliminating the need for column purication of intermediates in these multistep syntheses.Both ALC-0315 1 and SM-102 6, two ionizable lipids being used in COVID-19 vaccine drug products, as well as FTT5 5, a relatively recent lipid-like compound which has seen  in vivo success, have been prepared by employing fatty aldehyde bisulte adducts, which allows for easy isolation of the resulting solid intermediates.Additionally, the bisulte adducts can be used directly in subsequent reductive amination reactions, in which they performed equally or better than the free aldehyde while also eliminating the need for CH 2 Cl 2 in the reaction.We believe this to be the rst reported isolation of solid fatty aldehyde bisulte adduct intermediates and their use in the production of ionizable lipids.The production of ALC-0315 1 via a solid fatty aldehyde bisulte adduct intermediate enabled an improved purication process to the typical column chromatography, improved yield when compared to other syntheses, and was found to perform equally when formulated with mRNA as other commercial sources of ALC-0315.Isolation of solid fatty aldehyde intermediates via bisulte adduct formation and ltration is viewed as a general approach to improve synthesis of other lipids and the isolation and purication of other fatty alkyl aldehyde intermediates.